The present invention relates to the field of human gene therapy, more in particular to gene therapy vehicles for the treatment of cardiovascular disease.
Hypertension and hypercholesterolemia are two of the main risk factors for human health in the Western world; these conditions can lead to atherosclerosis. Atherosclerosis may result in a number of severe cardiovascular diseases, like chronic heart failure, angina pectoris, claudicatio intermittens, or peripheral and myocardial ischemia. At least the early phases of atherosclerosis are characterised by endothelial dysfunction. Endothelial dysfunction causes coronary arterial construction, plays a role in both hypertension and hypercholesterolemia. It is one of the first measurable steps in the cascade of reactions leading to atherosclerosis, even before macroscopic lesions are evident. Many therapies have been investigated to assess the possibility to reverse the endothelial dysfunction, and to stimulate the formation of new blood vessels (angiogenesis). It has been suggested that oral L-arginine supplementation in the diet may be a therapeutic strategy to improve angiogenesis in patients with endothelial dysfunction.
It is well established that angiogenesis is mediated by a multitude of cytokines (like TNF-xcex1 and E-selectin) and angiogenic factors including bFGF (basic Fibroblast Growth Factor), VEGF (Vascular Endothelial Growth Factor), and TGF-xcex2. Both bFGF and VEGF are key regulators of angiogenesis in adult tissues. They selectively stimulate proliferation of endothelial cells, starting with the binding of these growth factors to receptors present on the endothelial cell surface. Nitric oxide (NO) has been shown to play a role in this process. NO, originally identified as endothelium-derived relaxing factor, is an important endothelial vasoactive factor.
While both NO and angiogenic factors like bFGF and VEGF play a key role in the endothelial functions, their precise mode of action is not known. On the one hand, levels of angiogenic factors like bFGF and VEGF are increased in patients suffering from endothelial dysfunction. On the other hand, is the release of nitric oxide in vascular endothelial dysfunction often reduced. This reduced release may cause constriction of the coronary arteries and thus contribute to heart disease. It is postulated that patients suffering from endothelial dysfunction could benefit from therapies to increase new collateral blood vessel formation and/or therapies to increase vasodilation.
Many experimental gene therapies concentrate on the stimulation of angiogenesis, in patients suffering from endothelial dysfunction, through the addition of VGEF or bFGF. Though these experimental therapies may have some effect, the level of therapy induced angiogenesis is low, leading to a slow, if at all, recovery or enhancement of blood flow.
It has been demonstrated that NO is involved in VEGF-mediated proliferation of endothelial cells. Exposure of endothelial cells to VEGF was shown to lead to the activation of constitutive NO synthase (ceNOS) and the release of biologically active NO. The proliferation of cells by VEGF can be inhibited by specific NOS-inhibitors like L-NAME, indicating that NO is an essential mediator in the VEGF-induced cell proliferation and angiogenesis.
Likewise, the presence of bFGF can increase ceNOS protein levels and enzyme activity during healing of rat gastric ulcers. Here also, the healing was inhibited specifically by the NOS-inhibitor L-NAME. In transgenic mouse models, disruption of the endogenous ceNOS gene impaired angiogenesis (Murohara et al.). This could not be compensated by the administration of VEGF, showing the essential role for NO in growth factor mediated angiogenesis.
The art teaches that NO is a secondary signal in the angiogenic response of endothelial cells to growth factors like bFGF and VEGF, and that NO acts as a downstream mediator of angiogenesis. Furthermore, the art suggest that the expression of the ceNOS gene in endothelial cells is a result of the induction by the growth factors, leading to the release of biologically active NO.
However, despite the increase in the levels of angiogenic factors like bFGF and VEGF, this does not result in sufficient collateral forming capacity.
In one aspect of the present invention we demonstrate that at least one of the limiting factors is NO. In another aspect of the invention we demonstrate that NO levels in the arterial wall are insufficient. In another aspect the invention provides a method for increasing angiogenesis through locally increasing NO and/or endothelial growth factors such as but not limited to VGEF and/or bFGF. In yet another aspect of the invention provides a method for increasing vasodilation of blood vessels. In another aspect the invention provides a method for increasing angiogenesis through locally delivering an expression vector, preferably an adenovirus vector, comprising at least an expression cassette for ceNOS, to sites selected for being provided with the capacity to induce, or at least in part promote, angiogenesis.